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Abstract

Recent evidence revealed that DNA is beyond just the blueprint of life; it is also involved in
immunomodulation. Unmethylated Cytosine-phosphate-Guanine (CpG) motifs of prokaryotic DNA
stimulate immune response by interacting with Toll-like receptor 9 (TLR9). This interaction is
mimicked using synthetic oligodeoxynucleotides (ODN) bearing similar DNA motifs to boost vaccinedriven
immune response in human. Conversely, mammalian telomeric ends expressing TTAGGG
repeats suppress immune response and contribute to fine-tuning of delicate immune balance. In this
respect, suppressive ODN A151 with such G-rich telomeric repeats has proven useful in
downregulating immune response; an overly active immune response is just as harmful to the host,
as in the case of autoimmune disorders. Both CpG ODN and A151 are currently under
preclinical/clinical trials with the aim of averting or medically treating a wide range of conditions
from cancer to infectious disease or from autoimmune to autoinflammatory conditions. Contrary to
CpG ODN, A151 literature is very limited and its modus operandi at gene level remains more of a
mystery. Additionally, the degree, duration and breath of A151-induced alterations in immune
transcriptome appear partially understood. Given the medical potential A151 holds for
immunosuppressive therapy in human as a “self-molecule”, understanding the underlying molecular
mechanisms via which A151 operates is invaluable. Toward this end, we attempted to uncover the
unidentified features lying behind A151 ODNs immunosuppressive effects on immune cell
transcriptome using a combined analysis approach of microarray data in this thesis. We
demonstrated for the first time that A151 ODN deprives the cells energy by ceasing cellular uptake of
fundamental molecules into the immune cells after derailing the entire intracellular trafficking.
Putting it another way, A151 does not directly act on immune system cells but actually suffocates the
cells by messing with intracellular trafficking, thereby blocking cellular uptake of fundamental
molecules like glucose and glutamine. As such, immune suppression is just an indirect consequence of this larger cellular chaos. Our results indicated that this phenomenon occurs independent of CpG
ODN stimulation of the cells and in a timely manner. Most, if not all, regulators of intracellular
trafficking, vesicle signaling, and membrane protein transportation were found downregulated after
incubation of cells with A151 at a physiologically relevant concentration, as well, implying full-blown
entry to this intracellular turmoil at cellular level. The A151 effect on immune transcriptome was not
just restricted to setting off a chaos for intracellular dynamics; novel long non-coding RNAs (lncRNAs)
with immunometabolic activities were identified within the scope of this study among elements
potentially regulated by A151, such as Lncpint, Malat1 and H2-T10 just to name a few. The
involvement of lncRNAs in immune regulation is a well-documented phenomenon. Finally, our data
showed that as an epiphenomenon of the intracellular turmoil mentioned above A151 has a deep
impact in immune cells on mTOR network, the cardinal network of cellular energetics, growth,
proliferation, and survival. A major shift in expression profile of relevant genes, i.e. downregulation
of many activators of mTOR signaling along with core mTOR components, was validated on the
benchtop after different layers of experimental validation using a wide range of marker genes and
functional assays, reflecting A151’s ability to vastly shape dynamics of metabolism in favor of a
metabolically inert state in macrophages and in B-cells. This knowledge will expand the breadth of
A151 therapy in the clinics.